Aspartate-like immunoreactivity in primary afferent neurons

Neuroscience

Vol. 40, No. 3, pp. 673486,

Printed in GreatBritain

1991

0306-4522/91$3.00+ 0.00 Pergamon Pressplc 0 1991IBRO

ASPARTATE-LIKE IMMUNOREACTIVITY AFFERENT NEURONS

IN PRIMARY

D. J. TRACEY*~$ S. DE BIASI& K. FIEND* and A. RUSTIONI’ *Department

of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A. $Dipartimento di Fisiologia e Biochimica Generali, Sezione di Istologia e Anatomia umana, Universita degli Studi di Milano, Italy Abstract-There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia. Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy: the great majority of cells immunopositive for aspartate were small (IS-30 pm in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors. This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.

these increases were significant for glutamate but not Glutamate and aspartate have excitatory actions on for aspartate. 3o Cultured dorsal horn neurons were many neurons in the central nervous system9 (for excited by glutamate but not aspartate,2s~26although reviews see Refs 8, 18, 28, 50). Both these excitatory these experiments were carried out under conditions amino acids are major candidates as neurotransmitin which N-methyl-D-aspartate (NMDA) receptors ters released by primary afferents.45 Both glutamate were blocked. and aspartate are present in the dorsal roots and Recent development of antibodies against excitdorsal root ganglia’7*29and both excite interneurons atory amino acids4*23*33,34,49 has produced further eviin the superficial laminae of the dorsal hom.46,47The dence that elevated levels of glutamate are present in latter observation suggests that they may be involved certain groups of cell bodies in dorsal root ganglia.57 in transmission of nociceptive information. Further Battaglia and Rustioni’ recently showed that glutasupport for this idea comes from the elevated concenmate was localized predominantly in small somata trations of both glutamate and aspartate in the spinal cord as a result of noxious stimulation in vivo.48 and that it was colocalized with substance P in some cells, lending further support to the idea that glutaHowever, the evidence for glutamate as a neurotransreleased by nociceptive mitter at synapses made by primary afferents is mate is a neurotransmitter afferents. Glutamate has also been demonstrated in stronger than the evidence for aspartate. Thus, stimusynaptic terminals in the superficial laminae of the lation of dorsal roots in vitro increased the concendorsal hom,36,“@ and it has also been demonstrated tration of glutamate and aspartate in the superfusate; in the terminals of primary afferents, again co-localeviized with substance P. I* Immunocytochemical tCurrent address: School of Anatomy, University of New South Wales, P.O. Box 1, Kensington NSW 2033, dence for aspartate in primary afferents is much more Australia. restricted, though the presence of high levels of this ~To whom correspondence should be addressed. amino acid has been suggested recently in the dorsal kbbreviations: iP7, 2-amino-7-phosphonoheptanoate; homU and in small caliber axons in dorsal roots.62 DAB, 3,3’-diaminobenzidine; HRP, horseradish peroxiLight microscopical immunocytochemistry was dase; NAAG, N-acetyl aspartylglutamate; NMDA, Nused in this study to investigate the presence of methyl-D-aspartate; PBS, phosphate-buffered saline. 673

in cell bodies of primary afferents, to determine its distribution among these cells and to see whether it is co-localized with glutamate. The presence of aspartate in terminals in the superficial laminae of the dorsal horn was also investigated using pre- and post-embedding immunocytochemistry with the electron microscope. A preliminary report of these data has been published.” aspartate

EXPERIMENTAL PROCEDURES

Light microscopical immunocytochemistry

Twenty-five adult Sprague-Dawley rats were used in these experiments. Of these, 16 were used in pilot experiments aimed at optimizing the conditions for perfusion and sectioning. Quantitative data were taken from nine rats. Each of these animals was anesthetized with sodium pentobarbital (75 mg/kg, i.p.), the thorax was opened, and the animal was then perfused with 5OOml filtered 4% cyanamide (Sigma C-2388) in phosphate buffer (pH 7.2), followed by SOOml filtered 4% paraformaldehyde in phosphate buffer at pH 7.2.’ Dorsal root ganglia from C5 to Tl were removed and post-fixed overnight in 4% paraformaldehyde. The meninges were removed, and the ganglia were transferred first to 0.1 M phosphate buffer (pH 7.2) and then to 30% sucrose in phosphate buffer (pH 7.2) before sectioning at 6 pm on a cryostat. In some experiments, paraffin sections were cut; ganglia were dehydrated, embedded in paraffin and serially sectioned at 4pm before mounting on glass slides and dewaxed. Staining procedures. Sections were rinsed 3 x 5 min in phosphate-buffered saline (PBS, pH 7.2) and incubated in 10% normal goat serum in PBS for 30min to mask nonspecific absorption sites. Sections were then treated overnight on the slide with drops containing primary antibody raised in rabbits against L-aspartate (484a2) conjugated to keyhole-limpet hemocyanin2’ The aspartate antiserum was normally diluted 1:5,000 in PBS for cryostat sections. In order to examine whether aspartate was colocalized with glutamate, alternate sections were treated with anti-glutamate (482a2, see Ref. 23; 645a2, supplied by Dr P. Petrusz). Glutamate antisera were diluted 1: 20,00& 1:40,000 in PBS for cryostat sections, and 1: 10,000 for paraffin sections. Drops containing c. 40 ~1 were applied to each section, and slides were kept overnight at 4°C in a humid environment. Sections were then rinsed 3 x 5 min in PBS, incubated again in 10% normal goat serum in PBS, and treated with biotinylated goat anti-rabbit IgG (1: 200 in PBS). Following this, they were rinsed 3 x 5 min in PBS and treated for 1 h with avidin-biotin-horseradish peroxidase (-HRP) complex at room temperature (Vector ABC; 10 ~1 biotinylated HRP in 1 ml of PBS). The sections were rinsed again 3 x 5 min in PBS, and incubated in 3,3’-diaminobenzidine (DAB) and hydrogen peroxide in cacodylate buffer (25mg DAB in 28.5 ml H,O + 1.5 ml 1N acetic acid + 20 ml Na cacodylate + 84 ~13% H,O,). The sections were then dehydrated, cleared and coverslipped and examined under a light microscope. Specificity of the antibodies used was tested in the dorsal root ganglia by carrying out blocking experiments. In these experiments, the primary antibody was preabsorbed either with its own antigen, or with other amino acids or peptides. We could then measure the extent to which staining was blocked by increasing concentrations of the blocking agent. Sections of a dorsal root ganglion (usually C7) were incubated with a mixture of the primary antibody and one of several blocking agents, including L-aspartate, r_-glutamate and N-acetyl aspartylglutamate (NAAG). In addition, two non-endogenous blocking agents, NMDA, and 2-amino-7phosphonoheptanoate (AP7) were used as part of an ongoing study to examine the possible relationship between

neuronal NMDA receptor sites and antibody bmding sites for glutamate and aspartate. 55 The primary antisera were used at the concentrations at which optimal staining had previously been found in pilot studies (see above). Control sections were incubated in primary antibody with no blocking agent, and in normal goat serum. Blocking agents (diluted in 0.01 M phosphate buffer in 0.9% saline, pH 7.2: PBS) were used at final concentrations of 0.01. 0. I. 1 and 1OmM. Video microscopy. Staining intensity and area were measured in individual neurons using video microscopy. Images were collected using a video camera (Hamamatsu C2400) connected to a high resolution frame-grabber (Data Translation DT2851) in an IBM PC/AT computer, and analysed quantitatively using proprietary software (Imagepro). This allowed us to measure the cross-sectional area and staining intensity of any given cell in a section. The resolution of the frame-grabber was eight bits, allowing 256 gray levels to be distinguished. Care was taken to adjust the parameters of the video amplifier such that all measured cells fell within this range, i.e. no measured cells were saturated black or white. For each pixel, the optical density was calculated from the gray value following calibration with a series of neutral density filters, using the logarithmic relation between gray value and optical density.‘4 .4n area of the slide free of sections was defined as having an optical density of zero. In some cases a digital shading correction was applied to reduce the unevenness of background illumination. The average optical density and equivalent diameter (the diameter of a circle with the same cross-sectional area as the cell) were thus derived for each measured neuron. Video microscopy allowed us to carry out three types of quantitative analysis. In tests for specificity of the antibodies used, staining intensity was measured at each concentration of each blocking agent. In a typical blocking experiment, a single dorsal root ganglion was sectioned on a cryostat to provide 22 slides, each carrying four 6-pm-thick sections. Sections on one slide were all treated with one concentration of one blocking agent. For each slide, a section was chosen and the density of staining of three representative small and relatively darkly staining neurons was measured. as was the density of three typical large and relatively lightly stained neurons, and three areas of background adjacent to the measured cells. Mean values were obtained, and staining intensity was then plotted against concentration of blocking agent (Figs 2, 3). Comparable experiments were also carried out on paraffin sections. We analysed the relation between cell diameter and intensity of staining for aspartate and glutamate. In five rats, cryostat sections of dorsal root ganglia were cut and stained for aspartate. For each animal, one ganglion was selected and the equivalent soma diameter and staining intensity were measured for each neuron in a population of 30.- 300 cells. Staining intensity was then plotted against soma diameter. In three rats, a similar analysis was carried out on neurons stained for glutamate. In these analyses, only those cells were measured in which the nucleus could be seen, in order to ensure that the diameter was measured close to its maximum value. Representative plots are shown in Fig. 5. Measurements were limited to sections on one slide, since different slides generally had slightly different levels of staining. We analysed the correlation between staining for aspartate and staining for glutamate in pairs of consecutive sections where one member of the pair was stained for aspartate, the second for glutamate. In one animal, a pair of sections from one DRG was chosen in which most cells could be identified in both sections, A camera lucida drawing was made of one of the two sections. Twenty cells were then identified on the drawing, and the staining intensity of each of these cells was measured for glutamate and aspartate. In a second animal, staining intensities were correlated for 52 cells. Correlation plots for these two animals are shown in Fig. 6.

Aspartate in primary afferent neurons

Electron microscopical immunocytochemistry Eight Sprague-Dawley rats were used in these experiments. Each of these animals was anesthetized with chloral hydrate or sodium pentobarbital (as above), the thorax was opened, and the animal was then perfused transcardially with 5OOml fixative. Cervical segments of the spinal cord were removed and postfixed for several hours. For pre-embedding immunocytochemistry, two rats were perfused with 2% cyanamide, 0.3% glutaraldehyde and 0.2% picric acid and postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). One rat was perfused with 2% glutaraldehyde and 0.5% paraformaldehyde and postfixed in the same fixative. Transverse sections of the spinal cord were cut on a Vibratome at 50 Hrn and incubated overnight at 4°C with the aspartate anti-serum (484a2; 1: 10.000 in PBS). Sections were then rinsed in PBS. incubated in 10% normal goat serum in PBS, treated with biotinylated goat anti-rabbit IgG (I:200 in PBS), rinsed 3 x 5 min in PBS and treated for 1 h with avidin-biotin-HBP complex at room temperature as de-

scribed above for sections of dorsal root ganglia. The spinal cord sections were then rinsed again 3 x 5 min in PBS, and incubated in DAB and hydrogen peroxide in cacodylate buffer. Selected sections were osmicated in 1% 0~0, for 1 h and wafer-embedded in Epon or Epon/Spurr’s. Ultrathin sections were cut from the superficial laminae of the dorsal horn, and examined under the electron microscope with or without counterstaining with uranyl acetate and lead citrate. For post-ern~~ng immunocytochemis~, five rats were perfused with 2.5% glu~raIdehyde and 0.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and postllxed in 4% paraformaldehyde. Transverse sections of the spinal cord were cut on a Vibratome, osmicated as above and wafer-embedded in Epon. Ultrathin sections were cut from the superticial laminae of the dorsal horn, collected on parlodion-coated slot nickel grids and etched for 30min with a saturated solution ofsodium meta~~~ate* followed by 1% sodium borohydride for 5 min. Immunogold staining was carried out using a modification of the method described by Varndell and Polak.% The primary antiserum against aspartate (484a2) was used at a concentration of 1: 3000-1:5000, and detected using colloidal gold particles (15-20 nm diameter) coated with goat anti-rabbit IgCi. Both the primary antiserum and the goat anti-rabbit IgG gold probe were applied for 1 h at room temperature. Immuno-

cytochemical controls included omission of the primary antiserum, replacement of the primary antiserum by normal rabbit serum, and incubation with the immunogold probe alone. RESULTS

Blocking experiments Aspurtate. In order to examine the specificity of the aspartate antibody in the dorsal root ganglia, cryostat sections and paraffin-embedded sections were incubated with primary antibody (1: 5ooOfor cryostat sections, 1: loo0 for paraffin sections) preabsorbed with one of the following agents: r.+-aspartate, L-glutamate, NAAG, or NMDA.s5 In the paraffin material, where a concentration of the primary antibody of at least 1: 1000 had to be used in order to obtain staining, neither aspartate nor other agents blocked staining and the effect of L-aspartate was similar to that of other agents. It was concluded that at this concentration, the aspartate antibody was crossreacting with other antigens in the tissue. In the cryostat material, with a concentration of 1: 5000, pre-absorption of the primary antibody

675

against aspartate with its own antigen showed that staining of small cells was blocked in a dose-dependent fashion (Figs 1, 2A). Pre-incubation with glutamate, NAAG or NMDA did not interfere with staining, except for slight suppression at a concentration of lOmM (Figs 1, 2B). This shows that in cryostat material, the aspartate antibody specifically recognizes aspartate in dorsal root ganglia and does not cross-react with those agents tested. Glutamate. The specificity of the two glutamate antibodies used was examined in cryostat sections of dorsal root ganglia. Sections were incubated with primary antibody at dilutions of 1: 60,000 (482a2) or 1:20,000 (645a2) containing one of the following agents: L-aspartate, L-glutamate, NAAG, NMDA or AP7. Pre-absorption of the primary antibodies with glutamate blocked staining of small cells in a concentration~e~ndent fashion (Fig. 3A). However, preincubation with aspartate, NAAG, NMDA and AP7 had no effect on staining of small cells at concentrations up to 1 mM, and did not reduce staining intensity below control values even at a concentration of 10mM (Pig. 33). This shows that in cryostat material, the glutamate antibody specifically recognizes glutamate in dorsal root ganglia and does not cross-react with those agents tested. Relation between cell size and staining intensity Staining intensity and contrast between lightly stained and darkly stained cells depended on the conditions of perfusion. Thus inclusion of glutaraldehyde in the fixative produced uniform dark staining in all neurons, and it appeared that the use of heparin as an anticoagulant prior to perfusion reduced the contrast between dark cells and light cells, particularly in material stained for glutamate. Aspartate. Immuno~ytochemical staining for aspartate in paraffin material was obtained only with 1: 500 and 1:lOOO dilutions of the antiserum. A subpopulation of small cells and no large cells were intensely stained. However, blocking experiments referred to above suggested that the aspartate antibody cross-reacted with glutamate at concentrations necessary to obtain staining in paraffin sections. When sections were cut on a cryostat, it was possible to stain cells using concentrations of the antibody of 1: 5000; at this concentration it could be shown that the antibody did not cross-react with glutamate (see above). The pattern of staining found in cryostat sections was comparable with that found in paraffin sections. The nuclear membrane was clearly visible, and unstained cells were often surrounded with stained satellite cells (Fig. 4A). A subpopulation of small cells was very darkly stained, while the remaining cells had staining intensities which varied from background levels (unst~ned) to moderately stained. In order to quantify the pattern of staining in dorsal root ganglion neurons, the relation between cell diameter and intensity of staining was examined. A representative scatter plot of

Fig. I. Photomicrographs to illustrate blocking of staining with the anti-aspartate antibody (484a2). A-C show three sections from one dorsal root ganglion (C7, R865), stained with anti-as~rtate (I : 5000). (A) Control section of dorsal root ganglion stained with anti-aspartate alone. (B) Section stained with anti-aspartate pre-absorbed with 10 mM aspartate. There is complete suppression of staining at this concentration. (C) Section stained with anti-aspartate pre-absorbed with 10 mM glutamate. There is slight suppression of staining at this concentration. Scale bar = 50pm.

Aspartate in primary afferent neurons 03

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Fig. 2. Specificity of the antibody against aspartate (484a2). The intensity of the stain produced by the antiserum is measured as optical density, using the video microscope and plotted against the concentration of the amino acid or peptide with which the antiserum has been pm-absorbed. (A) Intensity of staining (optical density) produced by anti-aspartate in a section of the dorsal root ganglion as the concentration of aspartate is increased from 0 mM (control). The upper plot (open squares) is for small, darkly stained neurons, the middle plot (diamonds) is for large, pale neurons while the lower plot (filled squares) shows measurements of background staining intensity. Each point represents the mean of three measurements (error bars represent SD). Note that staining intensity of small cell bodies decreases almost to background levels as the concentration of aspartate is increased to IOmM, suggesting that the aspartate antibody recognizes free aspartate. (B) Intensity of staining (optical density) produced by the anti-aspartate in small ceils, as the ~n~ntration of aspartate (open squares) and other agents (L-glutamate, filled diamonds; NAAG, tilled squares; and NMDA. open diamonds) is increased from 0 mM (control). While aspartate blocks staining of small dorsal root ganglion cells, the three other agents tested had little effect, although NMDA and glutamate reduced staining slightly below control levels at a concentration of 10 mM. This suggests that the antibody is specific to aspartate in the dorsal root ganglion.

staining intensity for aspartate against cell body size (equivalent diameter) is shown in Fig. 5A. The data shown are derived from a sample of 102 cells from

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cryostat sections of a single ganglion (R935); comparable results were obtained from five additional animals (R865, n = 248; R867, n = 43; R889, n = 29;

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Fig. 3. Specificity of the antibody against glutamate (645a2), measured as described for Fig. 2. (A) Intensity of staining (optical density) produced by the antibody against glutamate in a section of the dorsal root ganglion as the concentration of glutamate is increased from OmM (control). The upper plot (open squares) is for small, darkly stained neurons, the middle plot (filled squares) is for large, pale neurons while the lower plot (diamonds) shows measurements of background staining intensity. Each point represents the mean of three measutements (error bars represent S.D.). Note that staining intensity of small cell bodies decreases almost to background levels as the concentration of glutamate is increased to 10 mM, suggesting that the glutamate antibody recognizes free glutamate. The increase in staining seen at 0.01 mM is probably due to removal of non-specific antibodies from the polyclonal antibody by the blocking agents. These data suggest that the antibody is specific to glutamate in the dorsal root ganglion. (B) Intensity of staining produced by the antibody against glutamate (optical density) in small ceils, as the concentration of glutamate and other agents (L-glutamate, NAAG and NMDA) is increased from 0 mM (control). While glutamate blocks staining of small dorsal root ganglion cells, the three other agents tested had no effect at concentrations up to 1mM. At a concentration of 10 mM, NAAG and L-aspartate appear to inhibit staining but do not reduce it below control values.

Fig. 4. Photomicrographs to illustrate the staining pattern produced in sections of the dorsdi root ganglion by antibodies against aspartate and glutamate. (A) Cryostat section of C7 ganglion (R865) stained using anti-aspartate (484a2; l:SOOO). Note that some of the small cells are intensely stained. Differential interference contrast optics. Scale bar = 50 pm. (B, C) Co-localization of aspartate and glutamate as shown in adjacent cryostat sections from C8 ganglion (R935) stained for aspartate (1: 5000: B) and glutamate (1:20,000; C). Corresponding sections of the same labeled neurons are indicated (arrows). Corresponding sections of an unlabeled neuron are also shown (arrowheads). Neurons which are stained by the anti-aspartate antibody are also stained by the anti-glutamate antibody, but not with equal intensity. Scale bar = 50 pm.

R898, n = 45; R931, n = 41). Approximately 35% of ceils were small, with equivalent diameters of 20-30 pm. Of these small cetls, 5-10% were intensely stained, with optical densities of 0.1-0.2 at the antibody concentration used here. Approximately 85% of small cells had staining intensities above background levels; 15% of small cells were regarded as

unstained. These cells had optical densities of less than 0.04 in the experiment shown in Fig. 4A. ~~u~~~~ie, In paraffin sections, immunocyt~hemicat staining of dorsal root ganglia with anti-glutamate showed a pattern similar to that previously reported.’ Most of the small cells (equivalent diameters of 20-30pm) were darkly stained while most of the

Aspartate .in primary afferent neurons

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Fig. 5. Relation between cell size and staining intensity. (A) Plot showing the relation between cell size (equivalent diameter) and staining intensity (optical density) for neurons in a dorsal root ganglion stained for aspartate in cryostat material (R935, C8, n = 102). A population of small cells (15-30 Mm in diameter) is darkly stained for aspartate; a few of these are intensely stained, with optical densities of ~-0.1. Occasional large cells (> 30 pm) are also stained for aspartate. The horizontal line divides cells into those considered stained (above line) and those considered unstained (below line). (B) Plot showing the relation between cell size (equivalent diameter) and staining intensity (optical density) for neurons in the same dorsal root ganglion stained for glutamate in cryostat material (n = 102). As in A, a population of small cells (15-30 pm in diameter) is darkly stained for glutamate.

large cells (3&50 grn) were pale. Occasional large cells were darkly stained. Scatter plots of staining intensity for glutamate against cell body size (equivalent diameter) were constructed for paraffin-embedded material taken from the C7 ganglion of one animal (R860, n = 105). The scatter plot (not shown) showed that approximately 30% of measured cells were darkly stained. [Scatter plots were also constructed for S2 and S3 ganglia in one animal (courtesy of Dr Bruce Gynther; R227, n = 104). These scatter plots showed a similar pattern to cryostat material.] In cryostat sections, the best results were obtained with antibody 645a2. The results were generally similar to those derived from paraffin-embedded material, except that a smaller proportion of cells was darkly stained (c. 15%). Remaining cells had staining intensities which varied from background (unstained) to moderate; no large cells were darkly stained. The nuclear membrane was usually not visible. Large, unstained cells were often surrounded by a ring of stained satellite cells. A representative scatter plot of staining intensity for glutamate against cell body size is shown in Fig. 5B (R935, n = 102). Cells with optical densities of less than 0.03 were regarded as unstained in this experiment. Comparable data were found in two additional animals where staining was satisfactory (R898, n = 39; R931, n = 47). Co-localization

of aspartate

and glutamate

Once it had been established by blocking experiments that both anti-aspartate and anti-glutamate were specific for their antigens, we could attempt to answer the question as to whether aspartate and glutamate were co-localized in the same cell. The first technique used was to take a pair of adjacent cryostat sections of a dorsal root ganglion,

one stained with anti-glutamate and the other with anti-aspartate. The first section was then drawn under the camera lucida, and this drawing was then matched with an adjacent section stained with antiaspartate. From this it became clear that most cells which stain heavily for aspartate also stain heavily for glutamate (Fig. 4B, C). A difficulty with this “matching” technique is that there is a range of staining intensities, as can be seen in the scatter plots of cell size vs staining intensity (Fig. 5). To classify a given cell as “stained” or “unstained” then becomes an arbitrary decision. To avoid this problem, a quantitative technique of examining co-localization was devised. In each of three animals, a pair of adjacent cryostat sections of a dorsal root ganglion was taken, the first stained with anti-glutamate and the second with anti-aspartate. In each ganglion, a series of cells was chosen which could be identified in both sections. The staining intensity of each cell for both glutamate and aspartate was then quantified using the video microscope, and for each cell the staining intensity for glutamate was plotted against staining intensity for aspartate. When staining for glutamate was correlated for staining with aspartate, correlation coefficients (r) of 0.6-0.9 were found in three animals (R868, n = 25, r =0.83; R889, n = 20, r = 0.91; R935, n = 52, r = 0.65). The correlation between staining intensity for aspartate and glutamate for two of these animals is shown in Fig. 6. In control experiments, a similar procedure was carried out on adjacent sections stained with the same antibody. Under these circumstances, under ideal conditions there should be a perfect correlation between staining intensities of matched cells, i.e. a correlation coefficient of 1.0. In these controls

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Fig. 6. Correlation of staining intensity for aspartate and glutamate. Each plot shows a correlation of the staining intensity for aspartate and glutamate for a series of individual cells from one ganglion. Each point on the plot represents a single cell. A shows data for 20 cells (R889). The cluster of cells in the lower left quadrant is unstained for glutamate and aspartate; cells in the upper right quadrant are stained for both

glutamate and aspartate. There is a high correlation between staining intensity for glutamate (x-axis) and staining intensity for aspartate (y-axis); the correlation coefficient (r) is 0.91. B shows data for 52 cells from another animal (R935). Here the same trend is apparent, although r is lower (0.65). (R868), correlation coefficients of >0.9 were found when aspartate staining was correlated with aspartate staining or glutamate staining correlated with glutamate staining. These results suggest that the staining intensity and probably the concentration of glutamate and aspartate in cells of the dorsal root ganglia are strongly but not perfectly correlated. Presence of aspartate in terminals Thin sections of the superficial laminae of the spinal cord were examined using pre- and post-embedding immunocytochemistry. This revealed two types of terminals labeled by the aspartate antibody: small, dome-shaped terminals and large scalloped terminals. Both of these terminals made asymmetric synaptic contacts. The dome-shaped terminals contained many small, clear synaptic vesicles and occasional dense core vesicles; they generally made a single synaptic contact with small to medium sized dendrites. Labeled scalloped terminals were of two types: dark and light terminals. The dark scalloped terminals (Type I) had a relatively electron-dense cytoplasm, a very sinuous profile and contacted several postsynaptic elements. They contained many clear vesicles, heterogeneous in size with some very large, and occasional dense core vesicles. Light scalloped terminals (Type II) had a less sinuous profile than dark scalloped terminals. They contacted several postsynaptic elements and contained exclusively clear vesicles, homogeneous in size. They contained many mitochondria and occasional neurofilaments. Preembedding immunocytochemistry labeled both types of scalloped terminals; in lightly labeled terminals reaction product appeared to coat mostly the outer surface of synaptic vesicles, while in terminals which were more heavily labeled the reaction product appeared to spread through the terminal, but never

inside the vesicles or other organelles (Fig. 7A). With post-embedding immunocytochemistry, labeling appeared to be specific for some scalloped terminals (both dark, Fig. 7B, and light, Fig. 7C, D) as judged by the observation of a higher than background density of gold particles over these terminals (Fig. 8) and by the presence of adjacent unlabelled vesiclecontaining profiles. Although gold particles were located over terminals, they did not appear to be preferentially located over synaptic vesicles. Approximately 50% of dark scalloped terminals and 30% of light scalloped terminals showed aspartate-like immunoreactivity.

DISCUSSION

The main conclusions of this study were that (1) aspartate-like immunoreactivity can be demonstrated in a population of small ceil bodies in the dorsal root ganglia, (2) aspartate and glutamate are generally co-localized in these cell bodies and (3) aspartate is localized in synaptic terminals in the superficial laminae of the dorsal horn. Before discussing these conclusions below we will address some technical issues. Fixation It was clear from pilot studies and from previous work’ that the pattern of staining was critically dependent on the means of fixation, particularly for glutamate. Part of the reason appears to be that excitatory amino acids act not only as neurotransmitters, but are involved in numerous metabolic pathways including those for the synthesis and degradation of other amino acids.3.‘9 It may be that when contrast between stained and unstained cells is detectable, “metabolic” aspartate and glutamate have been released from cell bodies in the dorsal root

Fig. 7. Electron microscopic immunocytochemistry of terminals in the dorsal horn using anti-aspartate. A is from material processed for pre-embedding immunocytochemistry and shows three intensely labeled scalloped terminals (arrows) identifiable as dark (type I) due to the presence of very large clear vesicles and sinuous profiles. Note adjacent unlabeled boutons. B-D are from material processed for post-embedding immunocyt~h~stry using 15 nm (B, C) or 2Onm (D) goid particles. B shows a labeled dark scalloped terminal {arrow). C shows a light scalloped terminal (arrow) rich in small clear vesicles and mitochondria. Adjacent profiles are unlabeled. D shows another light scalloped terminal (arrow) rich in small clear vesicles and mitochondria. Scale bar = 0.5 hrn. 681

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study, which used unconjugated amino acids and other agents to show that the antibodies are specific under the conditions in which they have been used. Thus staining of cell bodies by anti-aspartate was blocked by pre-absorption with L-aspartate, but not by pre-absorption with L-glutamate or N A A G in concentrations less than 10 mM. In the same way, staining of cell bodies by anti-glutamate was blocked by pre-absorption with L-glutamate, but not by preabsorption with L-aspartate or NAAG. In view of this, it seems likely that the antibodies against aspartate and glutamate are specific to their respective amino acids, and are not recognizing some endogenous dipeptide or other substance.

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(39) (40) (75) (36) (3o) Fig. 8. Values represent number of gold particles//t m 2+ S.E of the mean (S.E.M.) over different profiles from lamina II of the dorsal horn. S, scalloped terminals; F, terminals with flat vesicles and forming symmetric synapses; D, dendrites; G, glia; B, background (vessel lumen or myelin sheath). The number of profiles from which counts were taken is shown in parentheses at bottom. S profiles (black bar) had significantly higher density of gold particles than each of the other profiles as evaluated by two-tailed t-test (P < 0.001, corrected to adjust for multiple comparisons). Values are from two pooled experiments. ganglia (and possibly taken up by satellite cells16,58) while the "transmitter" pool in the cells is retained. In fact it appears that the metabolic pool of glutamate can be selectively depressed by incubation of brain slices in artificial cerebrospinal fluid before immersion fixation, and under these conditions there may be a loss of glutamate-like immunoreactivity from cell bodies and dendrites, while terminals retain their immunoreactivity. 42'5~Aspartate-like immunoreactivity behaves in a similar way. 5° Staining for both glutamate and aspartate required higher concentrations of antiserum in paraffin sections than in cryostat sections. This was presumably because the additional processing, including exposure to alcohol and xylene, reduced the antigenicity of the amino acid conjugates. Specificity

One of the most important factors on which these conclusions depend is the specificity of the antibodies. This has been demonstrated previously for antibodies 482a2 and 484a2, which were shown to be specific when tested against conjugates of 18 amino acids and dipeptides. 23 Other laboratories have also found these antibodies to be specific27'37'62and this was confirmed by the blocking experiments carried out in the current

Quantitative analysis of digital images proved a valuable technique in this study, but several precautions had to be taken to ensure that results were accurate and reproducible. These included avoidance of saturation, correction of uneven illumination, and conversion of gray values to optical density values. 24 This conversion was carried out since gray values depend on the gain and offset of the video amplifier used, while optical density values are independent of these settings and allowed direct comparison of data from different experiments. Other technical factors may have biased our quantitative data to some extent. One of these is the difficulty of identifying the nucleus in glutamate-stained sections, particularly in lightly stained cells. Since identification of a nucleus was necessary for inclusion of a cell in the data, the proportion of large cells immunoreactive for glutamate is probably underestimated. Presence o f aspartate in cell bodies

Most small cell bodies with equivalent diameters between 15 and 30 #m were labeled for aspartate in this study; these labeled cells formed about 15-30% of the whole population and about 85% of small cells. The cells which are positive for aspartate appear to correspond with cells classified as small clark neurons using other criteria, while cells negative for aspartate probably correspond with large light cells which label with an antibody (RT97) against neurofilament protein. 3j Cells negative for aspartate and positive for RT97 have a broad distribution of diameters. We are not aware of previous immunocytochemical demonstrations of aspartate in dorsal root ganglion cells. However, aspartate has recently been reported to be present in about 15% of unmyelinated axons and 4% of myelinated axons in the rat dorsal root. 62 Thus the proportion of unmyelinated fibers immunopositive for aspartate is markedly less than the proportion of small neurons found in our data, although both studies used the same antibody. This discrepancy may be due in part to reduction in antigenicity of the aspartate conjugate by the additional processing required to increase antibody penetration in the study of Westlund et alp 2

Aspartate in primary afferent neurons Co-localization of aspartate and glutamate

It has been known for some time that aspartate and glutamate are both present in dorsal root ganglia.17*29 The separate demonstrations that aspartate’j2 and glutamate6’ are present in some unmyelinated axons of the dorsal root raises the question of the extent to which the two amino acids are co-localized in primary afferent neurons. Results of the present study suggest that the extent of co-localization is very high; no cells were found which were immunopositive for aspartate but not glutamate, and staining intensities for the two amino acids were positively correlated. It is clear that aspartate and glutamate are closely related metabolically” such that aspartate and a-ketoglutarate are in equilibrium with glutamate and oxaloacetate, a conversion which is mediated by aspartate aminotransferase. Thus it would not be surprising to find the two amino acids co-localized in the same cells, although immunocytochemistry has so far revealed only low levels of aspartate aminotransferase in dorsal root ganglion cells.’ It is perhaps more surprising to find that some neurons in the hippocampus and cerebral cortex may contain high levels of glutamate but not aspartate, while others contain high levels of aspartate but not glutamate.7*‘5~2’~43 Thus co-localization of aspartate and glutamate is not a general rule. The small cell bodies of dorsal root ganglia contain not only aspartate and glutamate, but also a number of peptides which may serve as neurotransmitters or neuromodulators. Substance P is found in some small cell bodies and is co-localized with glutamate in about 15% of small dorsal root ganglion neurons.’ Other peptides which have been reported include calcitonin gene-related peptide, cholecystokinin, dynorphin, [Leulenkephalin and somatostatin (see Ref. 10 for review). Peptides such as substance P may act as neuromodulators and produce slow depolarization of dorsal horn neurons.53 They may also be involved in specifying connections between small primary afferents and different types of target tissue such as skin, muscle and joint.20.4’ However, it seems likely that fast synaptic transmission from primary afferents to second-order neurons in the dorsal horn is mediated by excitatory amino acids rather than by peptides. Presence of aspartate in synaptic terminals

Two main types of terminal were found labeled with the aspartate antibody in the superficial laminae of the dorsal horn; dome-shaped terminals and scalloped terminals. Dome-shaped terminals may belong to local interneurons or to the axons of descending pathways such as the corticospinal tract. Scalloped terminals are believed to be associated with the axons of primary afferents; of these, the light terminals have been linked to myelinated fibers, and the dark terminals are believed to belong to unmyelinated fibers in the rat.6 In previous work it was shown that some dark scalloped terminals were of primary afferent

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origin and were immunopositive for g1utamate.‘2 Preliminary data suggest that aspartate and glutamate are co-localized in some scalloped terminals, but that co-localization is not complete-approximately 70% of dark scalloped terminals are immunopositive for glutamate12 but only about 50% are immunopositive for aspartate. Role of aspartate as a neurotransmitter

It has been suggested that perhaps 50% of glutamate in the central nervous system is related to general metabolism of neurons, while 20-30% belongs to the neurotransmitter pool; unfortunately, comparable figures are not available for aspartate.” Is the presence of excitatory amino acids in small cell bodies of the dorsal root ganglia related to a metabolic pool or to a transmitter pool? The fact that these amino acids are found in a specific group of cells and terminals argues against a general metabolic role. It has been shown that cytochrome oxidase, a mitochondrial enzyme which has been used as a metabolic marker, is present in dorsal root ganglion cells of various sizes63and is not correlated with labeling for glutamate.’ This observation, which was repeated in this study (not reported in Results) suggests that aspartate and glutamate do not serve a purely metabolic function in the cell bodies of primary afferents. Again, the fact that labeling is selective for morphologically distinguishable types of terminals seems more likely to be due to differences in neurotransmitter than to differences in degrees of metabolic activity. The low density of cytochrome oxidase reaction product in the superficial laminae, relative to the rest of the spinal gray,63 also argues against the possibility that the labeling of synaptic terminals in these laminae is related to high metabolic activity. As mentioned above, aspartate and glutamate are closely linked metabolically, and the mechanism of uptake into terminals or synaptosomes for the two amino acids seems to be identical.“‘4 However, there are differences between aspartate and glutamate which have led to the suggestion that glutamate is more likely to be a neurotransmitter in primary afferents than aspartate. Apparent evidence against aspartate as a transmitter released by primary afferents was provided by Jahr and Jessel,24 who found that cultured dorsal horn neurons with inputs from primary afferents were excited by glutamate but not aspartate. However, these experiments were carried out under conditions of high [Mg2+] where NMDA receptors were blocked, and there is some evidence that aspartate is relatively specific for NMDA recptors, particularly at low concentrations.32,35 In the cerebral cortex, L-glutamate but not L-aspartate is taken up into synaptic vesicles38,39and released in a Ca2+-dependent fashion from synaptosomes.“” However, some slice preparations do show Ca2+-dependent release of L-aspartate,52 and it is possible that Ca2+-dependent aspartate release from vesicles is

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particularly labile.4” On balance it seems quite likely that aspartate and glutamate are both released from the terminals of small diameter primary afferents, with aspartate having a relatively specific action on NMDA receptors while glutamate affects both NMDA and non-NMDA receptors. The neurons of the dorsal root ganglia which contain aspartate and glutamate are mainly of the small dark type (see above) and these are associated with afferent fibers with low conduction velocities in the C-fiber range’* so that many are associated with nociceptive endings. Evidence that aspartatc may function as a neurotransmitter reIeased by small diameter primary afferents includes the finding that aspartate levels are increased in the spinal cord superfusate following noxious stimulation.4H Nowever, these increases could be due to release from intemeurons. More persuasive is the evidence that aspartate (like glutamate) tends to excite those inter-

Pt al

neurons in the superficial laminae of the dorsal horn which are activated by C-tibers,4h~4’ CONCLUSION

In summary, we have evidence that aspartate and glutamate are present in small cell bodies of primary afferents, and that central terminals of unmyehnated or thinly myelinated fibers contain aspartate. Since primary afferents with small cell bodies and unmyelinated or thinly myelinated axons are associated with nociceptors, it is likely that both aspartate and glutamate are released by the central terminals of nociceptive primary afferents in the superficial lam&e of the dorsal horn. Acknowledgements-We would like to thank Dr Richard Weinberg for invaluable suggestions at all stages of this study, Mr Paul Johnson for software development. and Dr Peter Petrusz for constructive discussions.

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29 June 1990)